scholarly journals Nucleocytoplasmic Trafficking of G2/M Regulators in Yeast

2008 ◽  
Vol 19 (9) ◽  
pp. 4006-4018 ◽  
Author(s):  
Mignon A. Keaton ◽  
Lee Szkotnicki ◽  
Aron R. Marquitz ◽  
Jake Harrison ◽  
Trevin R. Zyla ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Cell Cycle Regulators ◽  
Cyclin Dependent Kinase ◽  
Nucleocytoplasmic Trafficking ◽  
Nucleocytoplasmic Shuttling ◽  
Upstream Regulator ◽  
Cellular Compartments ◽  
Cdk Regulation

Nucleocytoplasmic shuttling is prevalent among many cell cycle regulators controlling the G2/M transition. Shuttling of cyclin/cyclin-dependent kinase (CDK) complexes is thought to provide access to substrates stably located in either compartment. Because cyclin/CDK shuttles between cellular compartments, an upstream regulator that is fixed in one compartment could in principle affect the entire cyclin/CDK pool. Alternatively, the regulators themselves may need to shuttle to effectively regulate their moving target. Here, we identify localization motifs in the budding yeast Swe1p (Wee1) and Mih1p (Cdc25) cell cycle regulators. Replacement of endogenous Swe1p or Mih1p with mutants impaired in nuclear import or export revealed that the nuclear pools of Swe1p and Mih1p were more effective in CDK regulation than were the cytoplasmic pools. Nevertheless, shuttling of cyclin/CDK complexes was sufficiently rapid to coordinate nuclear and cytoplasmic events even when Swe1p or Mih1p were restricted to one compartment. Additionally, we found that Swe1p nuclear export was important for its degradation. Because Swe1p degradation is regulated by cytoskeletal stress, shuttling of Swe1p between nucleus and cytoplasm serves to couple cytoplasmic stress to nuclear cyclin/CDK inhibition.

scholarly journals Nucleocytoplasmic Shuttling of the Cdc42p Exchange Factor Cdc24p

2000 ◽  
Vol 148 (6) ◽  
pp. 1115-1122 ◽  
Author(s):  
Aljoscha Nern ◽  
Robert A. Arkowitz
Keyword(s):  
Cell Cycle ◽  
Actin Cytoskeleton ◽  
Nuclear Import ◽  
Vegetative Growth ◽  
Nuclear Export ◽  
Nuclear Accumulation ◽  
Nucleocytoplasmic Shuttling ◽  
Exchange Factor ◽  
Cycle Dependent ◽  
Necessary And Sufficient

Cdc24p, the GDP/GTP exchange factor for the regulator of actin cytoskeleton Cdc42p, localizes to sites of polarized growth. Here we show that Cdc24p shuttles in and out of the yeast nucleus during vegetative growth. Far1p is necessary and sufficient for nuclear accumulation of Cdc24p, suggesting that its nuclear import occurs via an association with Far1p. Nuclear export is triggered either by entry into the cell cycle or by mating pheromone. As Far1p is degraded upon entry into the cell cycle, cell cycle–dependent export of Cdc24p occurs in the absence of Far1p, whereas during mating similar export kinetics indicate that a Cdc24p–Far1p complex is exported. Our results suggest that the nucleus serves as a store of preformed Cdc24p–Far1p complex which is required for chemotropism.

scholarly journals Distinct Subcellular Localization Patterns Contribute to Functional Specificity of the Cln2 and Cln3 Cyclins of Saccharomyces cerevisiae

2000 ◽  
Vol 20 (2) ◽  
pp. 542-555 ◽  
Author(s):  
Mary E. Miller ◽  
Frederick R. Cross
Keyword(s):  
Cell Cycle ◽  
Subcellular Localization ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Cytoplasmic Localization ◽  
Cyclin Dependent Kinase ◽  
Functional Profile ◽  
Functional Specificity ◽  
Biochemical Fractionation ◽  
Nuclear Events

ABSTRACT The G1 cyclins of budding yeast drive cell cycle initiation by different mechanisms, but the molecular basis of their specificity is unknown. Here we test the hypothesis that the functional specificity of G1 cyclins is due to differential subcellular localization. As shown by indirect immunofluorescence and biochemical fractionation, Cln3p localization appears to be primarily nuclear, with the most obvious accumulation of Cln3p to the nuclei of large budded cells. In contrast, Cln2p localizes to the cytoplasm. We were able to shift localization patterns of truncated Cln3p by the addition of nuclear localization and nuclear export signals, and we found that nuclear localization drives a Cln3p-like functional profile, while cytoplasmic localization leads to a partial shift to a Cln2p-like functional profile. Therefore, forcing Cln3p into a Cln2p-like cytoplasmic localization pattern partially alters the functional specificity of Cln3p toward that of Cln2p. These results suggest that there are CLN-dependent cytoplasmic and nuclear events important for cell cycle initiation. This is the first indication of a cytoplasmic function for a cyclin-dependent kinase. The data presented here support the idea that cyclin function is regulated at the level of subcellular localization and that subcellular localization contributes to the functional specificity of Cln2p and Cln3p.

scholarly journals Unconventional tonicity-regulated nuclear trafficking of NFAT5 mediated by KPNB1, XPOT and RUVBL2

2021 ◽  
Author(s):  
Chris Y. Cheung ◽  
Ting-Ting Huang ◽  
Ning Chow ◽  
Shuqi Zhang ◽  
Yanxiang Zhao ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Shuttling ◽  
Proteomics Analysis ◽  
Nuclear Trafficking ◽  
Localization Signal ◽  
Underlying Mechanisms ◽  
Sirna Screening

NFAT5 is the only known mammalian tonicity-responsive transcription factor functionally implicated in diverse physiological and pathological processes. NFAT5 activity is tightly regulated by extracellular tonicity but the underlying mechanisms remain elusive. We demonstrated that NFAT5 enters the nucleus via the nuclear pore complex. We also found that NFAT5 utilizes a non-canonical nuclear localization signal (NFAT5-NLS) for nuclear imports. siRNA screening revealed that karyopherin beta-1 (KPNB1) drives nuclear import of NFAT5 via directly interacting with NFAT5-NLS. Proteomics analysis and siRNA screening further revealed that nuclear export of NFAT5 under hypotonicity is mediated by Exportin-T, and that it requires RuvB-Like AAA type ATPase 2 (RUVBL2) as an indispensable chaperone. Our findings have identified KPNB1 and RUVBL2 as key molecules responsible for the unconventional tonicity-regulated nucleocytoplasmic shuttling of NFAT5. These findings offer an opportunity for developing novel NFAT5 targeting strategies that are potentially useful for the treatment of diseases associated with NFAT5 dysregulation.

Nucleocytoplasmic Shuttling of Porcine Parvovirus NS1 Protein Mediated by the CRM1 Nuclear Export Pathway and the Importin α/β Nuclear Import Pathway

2021 ◽  
Author(s):  
Liyan Cao ◽  
Fang Fu ◽  
Jianfei Chen ◽  
Hongyan Shi ◽  
Xin Zhang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Nonstructural Protein ◽  
Nuclear Pore ◽  
Porcine Parvovirus ◽  
Ns1 Protein ◽  
Nucleocytoplasmic Shuttling ◽  
Export Pathway ◽  
Importin Α

Porcine parvovirus (PPV) NS1, the major nonstructural protein of this virus, plays an important role in PPV replication. We show, for the first time, that NS1 dynamically shuttles between the nucleus and cytoplasm, although its subcellular localization is predominantly nuclear. NS1 contains two nuclear export signals (NESs) at amino acids 283–291 (designated NES2) and 602–608 (designated NES1). NES1 and NES2 are both functional and transferable NESs, and their nuclear export activity is blocked by leptomycin B (LMB), suggesting that the export of NS1 from the nucleus is dependent upon the chromosome region maintenance 1 (CRM1) pathway. Deletion and site-directed mutational analyses showed that NS1 contains a bipartite nuclear localization signal (NLS) at amino acids 256–274. Coimmunoprecipitation assays showed that NS1 interacts with importins α5 and α7 through its NLS. The overexpression of CRM1, importins α5 and α7 significantly promoted PPV replication, whereas the inhibition of CRM1 and importin α/β-mediated transport by specific inhibitors (LMB, importazole and ivermectin) clearly blocked PPV replication. The mutant viruses of delete NESs or NLS motif of the NS1 by using reverse genetics could not be rescued, suggesting that NESs and NLS are essential for PPV replication. Collectively, these findings suggest that NS1 shuttles between the nucleus and cytoplasm, mediated by its functional NESs and NLS, via the CRM1-dependent nuclear export pathway and the importin α/β-mediated nuclear import pathway, and PPV proliferation was inhibited if blocking NS1 nuclear import or export. Importance PPV replicates in the nucleus, and the nuclear envelope is a barrier to its entry into and egress from the nucleus. PPV NS1 is a nucleus-targeting protein that is important for viral DNA replication. Because the NS1 molecule is large (> 50 kDa), it cannot pass through the nuclear pore complex by diffusion alone, and requires specific transport receptors to permit its nucleocytoplasmic shuttling. In this study, the two functional NESs in the NS1 protein were identified, and its dependence on the CRM1 pathway for nuclear export demonstrated. The nuclear import of NS1 utilizes importins α5 and α7 in the importin α/β nuclear import pathway.

scholarly journals Active Nuclear Import and Export Is Independent of Lumenal Ca2+ Stores in Intact Mammalian Cells

1999 ◽  
Vol 113 (2) ◽  
pp. 239-248 ◽  
Author(s):  
Carsten Strübing ◽  
David E. Clapham
Keyword(s):  
Active Transport ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Protein Transport ◽  
Fluorescent Protein ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Trafficking ◽  
Acetoxymethyl Ester ◽  
Store Depletion

The nuclear pore complex (NPC) mediates communication between the cytoplasm and nucleus in eukaryotic cells. Active transport of large polypeptides as well as passive diffusion of smaller (≈10 kD) macromolecules through the NPC can be inhibited by depletion of intracellular Ca2+ stores. However, the physiological relevance of this process for the regulation of nucleocytoplasmic trafficking is not yet clear. We expressed green fluorescent protein (GFP)–tagged glucocorticoid receptor (GR) and mitogen-activated protein (MAP) kinase–activated protein kinase 2 (MK2) to study the effect of Ca2+ store depletion on active transport in HM1 cells, a human embryonic kidney cell line stably transfected with the muscarinic M1 receptor. Dexamethasone-induced nuclear import of GR-GFP and anisomycin-induced nuclear export of GFP-MK2 was monitored by confocal microscopy. We found that store depletion by carbachol, thapsigargin or ionomycin had no effect on GR-GFP import, whereas pretreatment with 1,2-bis-(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid–acetoxymethyl ester (BAPTA-AM) attenuated import significantly. Export of GFP-MK2 was not influenced by any pretreatment. Moreover, carbachol stimulated GFP-MK2 translocation to the cytoplasm in the absence of anisomycin. These results demonstrate that Ca2+ store depletion in intact HM1 cells is not directly linked to the inhibition of active protein transport through the NPC. The inhibition of GR-GFP import but not GFP-MK2 export by BAPTA-AM presumably involves a depletion-independent mechanism that interferes with components of the nuclear import pathway.

scholarly journals How SARS-CoV-2 and Other Viruses Build an Invasion Route to Hijack the Host Nucleocytoplasmic Trafficking System

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1424
Author(s):  
Elma Sakinatus Sajidah ◽  
Keesiang Lim ◽  
Richard W. Wong
Keyword(s):  
Nuclear Import ◽  
Nuclear Transport ◽  
Antiviral Treatment ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Trafficking ◽  
Nucleocytoplasmic Shuttling ◽  
Transport Receptors ◽  
Import And Export ◽  
Viral Components ◽  
Host Nucleus

The host nucleocytoplasmic trafficking system is often hijacked by viruses to accomplish their replication and to suppress the host immune response. Viruses encode many factors that interact with the host nuclear transport receptors (NTRs) and the nucleoporins of the nuclear pore complex (NPC) to access the host nucleus. In this review, we discuss the viral factors and the host factors involved in the nuclear import and export of viral components. As nucleocytoplasmic shuttling is vital for the replication of many viruses, we also review several drugs that target the host nuclear transport machinery and discuss their feasibility for use in antiviral treatment.

scholarly journals Cyclin-dependent kinase 2 (Cdk2) controls phosphatase-regulated signaling and function in platelets

2020 ◽  
Author(s):  
Paul R. Woods ◽  
Brian L. Hood ◽  
Sruti Shiva ◽  
Thomas P. Conrads ◽  
Sarah Suchko ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Platelet Activation ◽  
Human Platelets ◽  
Cell Cycle Regulators ◽  
Cyclin Dependent Kinase ◽  
Erk Activation ◽  
Src Signaling ◽  
Energetic State ◽  
And Function

AbstractCell cycle regulatory molecules including cyclin-dependent kinases can be recruited into non-nuclear pathways to coordinate cell cycling with the energetic state of the cell or with functions such as motility. Little is known about the role of cell cycle regulators in anucleate cells such as platelets. We report that cyclin-dependent kinase (cdk2) is robustly expressed in human platelets, is activated by thrombin and is required for platelet activation. Cdk2 activation required Src signaling downstream of the platelet thrombin receptor PAR1. Kinase-active cdk2 promoted the activation of downstream platelet kinases by phosphorylating and inactivating the catalytic subunit of protein phosphatase 1 (PP1). Erk was bound to PP1 in a complex with the PP1 regulator PPP1R12a (MYPT1) in platelets, and cdk2 inhibited the phosphatase activity of PP1 and PPP1R12a bound complexes. The requirement for cdk2 in Erk activation could be replaced by the phosphatase inhibitor calyculin if cdk2 was inhibited. Blockade of cdk2 kinase with chemical and peptide cdk2 inhibitors resulted in suppression of thrombin-induced platelet aggregation, and partially inhibited GPIIb/IIIa integrin activation as well as platelet secretion of P-Selectin and ATP. Together, these data indicate a requirement for cdk2 in platelet activation.

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